1. Field of the Invention
The present invention relates to a biologically active substance recovered from the mycelium and fruit body of Agaricus blazei; and a process of preparing the biologically active substance.
2. Description of the Prior Art
Agaricus blazei is a mushroom of Basidiomycetes from Brazil. It has been known that the mushroom is very effective for treating cancer and other adult diseases. In recent years, an artificial method for cultivating the mushroom has been developed, but because of the difficulty in the cultivation, currently, the supply is far lower than the growing demand.
Alternatively, active research works about Agaricus blazei have been promoted from the respect of medical and nutritional standpoint, reporting that the principally effective component of the mushroom is xcex2-glucan as one of the polysaccharides. xcex2-Glucan has such high immune activation potency that the glucan can activate immune cells (for example, natural killer cells) thereby attacking cancer cells and killing the cells. It has been known that xcex2-glucan is also present in mushrooms such as shiitake (Cortinellus shiitake) and maitake, other than Agaricus blazei. Renntinan extracted from shiitake as well as crestin extracted from kawaratake have been formulated as anti-cancer agents. Some of current research works have suggested that Agaricus blazei is a mushroom with the highest anti-cancer action.
Methods for extracting effective polysaccharides such as xcex2-glucan from Agaricus blazei have been proposed conventionally (for: example, Japanese Patent Disclosure No. 1-67195). Because the content of xcex2-glucan is very low in Agaricus blazei, however, a great amount of Agaricus blazei is required, to recover a given amount of the extract. Furthermore, Japanese Patent Disclosure No. 5-268905 describes a method for extracting extractable components from whole Agaricus blazei by using an enzymatic agent. By this method, an extract solution with flavor like matsutake (Armillaria matsutake) can be recovered, through the action of enzymatic agents containing endo-1,4-xcex2-glucanase, xylanase and endo-1,3-xcex2-glucanase on Agaricus blazei. 
By the method for extracting such extractable components, however, xcex2-glucan contained in the whole Agaricus blazei or xcex2-glucan recovered during the intermediate stage of treating the mushroom may further be decomposed to generate cellobiose and glucose, because the enzymatic agent containing xcex2-glucanase is utilized by the method.
It is an object of the present invention to recover a biologically active substance containing greater amounts of effective polysaccharides such as xcex2-glucan from the mycelium and fruit body of Agaricus blazei or the liquid waste derived from the culture of the mycelium. By utilizing hemicellulase in an enzymatic agent, in particular, an active polysaccharide containing a great amount of xcex2-glucan is recovered through the decomposition of hemicellulose as a structural sugar of Agaricus blazei, so that the yield of xcex2-glucan from Agaricus blazei can be improved.
So as to overcome the problems described above, the biologically active substance of the present invention characteristically contains a great amount of xcex2-glucan produced by decomposing and treating the mycelium or fruit body of Agaricus blazei or a liquid waste from the culture of the mycelium with an enzymatic agent principally, comprising hemicellulase.
The process of preparing the biologically active substance in accordance with the present invention comprises decomposing or treating the mycelium or fruit body of Agaricus blazei or a liquid waste from the culture of the mycelium with an enzymatic agent principally comprising hemicellulase.
The biologically active substance produced in accordance with the present invention contains a great amount of active low-molecular polysaccharides such as xcex2-D-glucan, and additionally contains effective ingredients such as nucleic acid other than the polysaccharides. The molecular weights of the active polysaccharides are about 2,000,000 to 500,000. By preparing the substance from such low molecular weight substances, the digestion and absorption thereof in the body are enhanced, so that the immune activation effect can be exerted.
The mycelium of Agaricus blazei can be prepared in liquid culture or solid culture. Because the liquid waste of the culture of the mycelium of Agaricus blazei other than the fruit body thereof contains xcex2-glucan, such biologically active substance can be recovered from the liquid waste.
The enzymatic agent to be used in accordance with the present invention principally comprises hemicellulase. As the hemicellulase of the present invention, use is made of enzyme groups (for example, mannase, arabinosidase, xylobiase, etc.) recovered from the culture of Tricoderma biride JAM 4033, Tricoderma haldianum JAM4031, Aspergillus tamari JAM 4007 and Aspergillus niger JAM 4012; and general enzymatic agents commercially available (for example, hemicellulase manufactured by Sigma, Co.). Hemicellulase may be used singly but may also be used after hemicellulase is mixed with pectinase, whereby the step-wise reaction of the enzyme treatment can be conducted in a smooth manner.
The preparation process of xcex2-glucan comprising enzymatically treating the mycelium thereby decomposing the mycelium is described in the schematic view of FIG. 1. Xylan, mannan and arabinan and the like in addition to xcex2-glucan are bound together to compose a long-chain fiber in the mycelium. By reacting an enzymatic agent in a mixture with hemicellulase or pectinase with the mycelium, hemicellulase is hydrolyzed in a step-wise manner, whereby the bonded chains therein are cut to prepare high-molecular polysaccharides, which are then converted to the active polysaccharide (xcex2-D-glucan).
The ratio of the enzymatic agent added to the mycelium is 0.01 to 0.5% by weight, preferably about 0.1% by weight. The enzymatic solution for the treatment is at pH 3.0 to 8.5, preferably about pH 4.5. The temperature for the enzymatic treatment is 25 to 60xc2x0 C., preferably about 45xc2x0 C. Furthermore, the duration for the enzymatic treatment is 20 to 120 minutes, preferably about 60 minutes.
Just when the reaction by enzymatic treatment progresses at some extent, the solution for the treatment is heated to terminate the enzyme reaction. The enzyme is generally inactivated by heating at 80 to 100xc2x0 C. for about 10 minutes. Through the termination of the enzyme reaction, the raw material of the active polysaccharide containing a greater amount of xcex2-glucan derived from Agaricus blazei is produced. By condensing and drying the material, the biologically active substance of the present invention is recovered. The drying process preferably comprises freeze-drying, but may possibly comprise spray drying because the effective ingredient is relatively resistant to heat. The biologically active substance of the present invention contains xcex1-glucan, xcex2-galactoglucan, proteinous glucan and the like, in addition to the principal component xcex2-glucan.
As has been described above, the mycelium or fruit body of Agaricus blazei or a liquid waste of the culture of the mycelium is decomposed and treated with the enzymatic agent principally comprising hemicellulase, to improve the yield of the active polysaccharide containing a greater amount of xcex2-glucan.
Because the concentration of the active polysaccharide can be increased, furthermore, the digestion and absorption of the biologically active substance of the present invention after the incorporation thereof into bodies is prominently elevated, so that the active substance can exert excellent immune activation effect together with the accelerated effect, on cancers and other adult diseases.
The present invention will now be described in more detail, with reference to attached drawings.